(1) First, we compared RNA expression of different PDE subtypes in rabbit SANC and ventricular myocytes (VM). Total RNA was reverse transcribed to generate complementary DNA (cDNA), and relative abundance of cDNA from 9 different PDE transcripts was measured with qPCR. PDE3A, PDE4B and PDE4D were the major PDE subtypes expressed in both rabbit SANC and VM. We verified expression of major PDE subtypes (PDE3A, PDE4A, PDE4B and PDE4D) at the protein level in the rabbit SA node and ventricle using western blot. Consistent with the qPCR data, PDE3A and PDE4A protein was more abundant in the rabbit ventricle than in SA node. There was comparable expression of PDE4B protein in the SA node and ventricle, while expression of PDE4D protein was more abundant in the SA node. The intracellular distributions of the most abundant isoforms of PDE3 (PDE3A), and PDE4 (PDE4B and PDE4D) in rabbit SANC were examined using immunostaining. PDE3A was detected both beneath sarcolemma and in a striated pattern within Z-lines of rabbit SANC, colocalized with the Z-line associated protein alfa-actinin. PDE3A co-localized with PDE4B beneath sarcolemma of SANC, while PDE4D co-localized with PDE3A in striated patterns inside SANC. Co-staining of PDE3A with SERCA or PLB antibodies showed that PDE3A co-localized with SERCA and PLB in SANC. Since PDE4D co-localized with PDE3A, PDE4D should be also in the proximity of major SR proteins SERCA and PLB. To test our first hypothesis, we used phosphorylation of phospholamban (PLB) at Ser16 site as a marker of cAMP/PKA-dependent protein phosphorylation in SANC. Specific PDE3 inhibitor, cilostamide (Cil, 0.3 mkmol/L), or a PDE4 inhibitor, rolipram (Rol, 2 mkmol/L), increased PLB phosphorylation by 20%, but the combination of Cil+Rol increased PLB phosphorylation by 110%, an effect similar to that (140%) produced by broad spectrum PDE inhibitor IBMX. L-type Ca2+ current (ICa,L) ensures LCR existence, providing Ca2+ available for pumping into SR. Inhibition of PDE3 or PDE4 alone increased the amplitude of ICa,L by 60% and 4%, respectively, while dual PDE3+PDE4 inhibition or IBMX increased ICa,L by 100%. Inhibition of PDE3 alone increased spontaneous SANC firing was by 20%, while inhibition of PDE4 alone had no effect on spontaneous firing. Dual PDE3+PDE4 inhibition, however, similar to IBMX increased the spontaneous SANC firing by 50%. This effect was due to a marked increase in the LCR number, size and decrease in the LCR period that predicted the concomitant decrease in the spontaneous cycle length. When RyR were disabled by ryanodine and LCRs were abolished, both IBMX and (Cil+Rol) failed to accelerate DD rate or increase SANC firing rate indicating key role of Ca2+ cycling for PDE-dependent control of spontaneous beating. We conclude that spontaneous SANC firing is regulated by dual PDE3+PDE4 activation, and a crucial role of PDE4 is unmasked only when PDE3 and PDE4 are concurrently inhibited. Thus, synergism of combined (PDE3+PDE4) activation in SANC suppresses basal cAMP/PKA-dependent PLB phosphorylation and reduces ICa,L amplitude to decrease RyR Ca2+ release, prolong the LCR period and limit the spontaneous SANC firing rate. (2) Spontaneous firing of freshly isolated rabbit SANC was markedly suppressed by selective PKC inhibitors GF109203X or calphostin C, and this was accompanied by suppression of SR Ca2+ cycling. Eventually LCRs ceased and spontaneous beating of SANC stopped. Specifically, GF109203X decreased the LCR size and number per each spontaneous cycle and increased the LCR period, i.e. the time from the prior AP-induced Ca2+ transient to the subsequent LCR. The PKC inhibition produced increase in the LCR period correlated with the increase in the spontaneous SANC cycle length. All effects of GF109203X were reversible upon washout. Since Ca2+ cycling in SANC is critically dependent on L-type Ca2+ current (ICa,L), which contributes to the AP upstroke and modulates the SR Ca2+ content, we studied effects of GF109203X and calphostin C on ICa,L. PKC inhibition by both inhibitors markedly suppressed ICa,L amplitude, strongly suggesting that modulation of ICa,L could be one of the major targets of basal PKC activation in rabbit SANC. PKC could be activated by ubiquitous enzyme phospholipase C (PLC), which plays a key role in Ca2+ signaling in numerous cell types. (3) Inhibition of PLC activation by U-73122, but not its inactive analog U-73343, stopped spontaneous firing of intact rabbit SANC, indicating a key role of basal PLC activation for cardiac pacemaking. PLC could be activated through multiple pathways, including the cAMP mediator Epac or activation of epidermal growth factor receptor (EGFR) or vascular endothelial growth factor receptor (VEGFR). Indeed, there was high basal EGFR and VEGFR activation in SANC, since inhibition of either EGFR by AG1478 or VEGFR by vatalanib suppressed LCRs in a time-dependent manner, i.e. decreased average LCRs size, number per each spontaneous cycle, prolonged the LCR period and increased the spontaneous SANC cycle length. Selective Epac1 or Epac2 inhibitors (CE3F4 or ESI-05, respectively) also markedly decreased spontaneous SANC beating rate. Specifically, both CE3F4 and ESI-05 decreased the LCR size and number per each spontaneous cycle, prolonged the LCR period and increased spontaneous cycle length, suggesting elevated basal activation of both Epac1 and Epac2 in rabbit SANC. Thus, basal activation of Epac1, Epac2, EGFR or VEGFR (and probably others) stimulates basal PLC activation and PLC-dependent control of spontaneous SANC firing, which operates through basal PKC activation and is obligatory for normal automaticity of cardiac pacemaker cells.